Transforming growth factor-β (TGF-β) is a 25 kDa multi functional growth factor which plays a central role in the wound healing process (Roberts and Sporn, 1990; O'Kane and Ferguson, 1997). It is an important regulator of the immune response (Letterio and Roberts, 1998), angiogenesis, reepithelialization (Roberts and Sporn, 1990), extracallular matrix protein synthesis and remodeling (Peltonen et al, 1991; Yamamoto et al, 1994). During wound healing, re-epithelialization initiates the repair process which is characterized by recruitment of epidermal stem cells, keratinocyte proliferation and the formation of an epithelial tongue of migrating keratinocytes at the wound edge (Clark, 1996). TGF-β is chemotacfic to keratinocytes and induces the expression of integrins on the migrating epithelium (Helbda, 1988; Zambruno et al, 1995). In spite of its promigratory effect on keratinocytes, TGF-β is a potent inhibitor to epithelial cell proliferation in vitro (Pietenpol et at, 1990) and in vivo (Glick et al, 1993). Targeted deletion of the TGF-β1 gene in keratinocytes causes rapid progression to squamous cell carcinoma (Glick et al, 1994). In addition, the epidermis of transgenic mice expressing a dominant negative TGF-β receptor exhibits a hyperplastic and hyperkeratotic phenotype (Wang et al, 1997). These results support the importance of proper expression of TGF-β and regulation of its function in epidermal development and maintenance of epidermal homeostasis.
TGF-β is a member of the TGF-β superfamily which also include activins, inhibins, bone morphogenic proteins, growth differentiation factor 1 (GDF-1) and glial-derived neurotropic growth factor (GDNF) (Kingsley, 1994).
There are three widely distributed TGF-β receptors, type I, type II and type III, all of which have been cloned (Roberts and Sporn, 1990; Massague, 1998). The types I and II receptors are both transmembrane serine/threonine kinases that are essential for TGF-β signal transduction. The type III receptor, also known as betaglycan, is a high molecular weight proteoglycan that is not required for signaling, but is believed to play a role in presenting the ligand to the type II receptor (Lopez-Casillas et al, 1993). Endoglin, is another TGF-β receptor predominantly expressed on endothelial cells (Gougos and Letarte, 1990). According to the present model of TGF-β signal transduction, binding of TGF-β to the type II receptor which is a constitutively active kinase, leads to the recruitment and phosphorylation of the type I receptor (Wrana et al, 1994). The activated type I kinase phosphorylates the central intracellular mediators of TGF-β signalling known as the Smad proteins (Heldin et al, 1997). Smad proteins can be classified into three groups: the pathway restricted Smads include the Smad2 and Smad3 which are phosphorylated by the type I receptor of TGF-β or activin, while the Smads 1, 5 and 8 are implicated in BMP signalling. The phosphorylation of the pathway restricted Smads permits their interaction with the common Smad or Smad4 and this heteromeric complex then translocates into the nucleus where it regulates expression of target genes. Finally, there inhibitory Smads which include the Smad 7 and Smad 6 prevent the phosphorlyation of the R-Smads by the type I kinase. (Heldin et al, 1997, Massague, 1998; Wrana and Attisano, 2000)
In blood circulation, TGF-β1 is found bound to the carrier α2 macroglobulin (α2M; Webb et al. 1998). α2M binds many other cytokines and therefore lacks selectivity for TGF-β1. α2M polymorphism has been associated with Alzheimer's disease, which polymorphism is observed as a deletion in “the bait region” overlapping with TGF-β1 binding domina (Gonias et al. 2000 and Blacker et al 1998).
Although the types I and II receptors are central to TGF-β signaling, it is possible that accessory receptors interacting with the signaling receptors modify TGF-β responses. For example, both endoglin and type III receptor which form heteromeric complexes with the type II receptor have been reported to modulate TGF-β function. When overexpressed in myoblasts, endoglin inhibited while type III receptor enhanced TGF-β responses (Letamendia et al, 1998). In addition, endoglin was shown to antagonize TGF-β mediated growth inhibition of human vascular endothelial cells (Li et al, 2000). Similarly, the newly identified type I-like receptor BAMBI which associates with TGF-β family receptors can inhibit signaling (Onichtchouk et al, 1999).
There are also a number of molecules that can impact TGF-β signal transduction by interacting with one or both of the TGF-β signaling receptors. However, the exact physiological significance of many of these interactions are not clearly defined (for review, Massague, 1998). Three of these interacting proteins: the type II TGF-β receptor interacting protein (TRIP-1) (Chen et al, 1995), Bα (α subunit of protein phosphatase A) (Griswold-Prenner, 1998), and serine-threonine kinase receptor associated protein (STRAP) (Datta et al, 1998) all contain the highly conserved tryptophan-aspartic acid (WD) repeats. WD domains are important in protein-protein interactions and cellular functions such as cell cycle progression and transmembrane signaling (Neer et al, 1994). TRIP-1 is phosphorylated through its interaction with the type II receptor kinase and exerts an inhibitory effect on TGF-β induced PAI-1 gene transcription, but has no effect on TGF-β mediated growth inhibition (Choy and Derynck, 1998). On the other hand, Bα associates with the type I receptor and positively modulates TGF-β action. Finally, STRAP can interact with both the type I and II receptors and when overexpressed, it exerts an inhibitory effect on TGF-β mediated transcriptional activation. In addition, STRAP can also interact with the inhibitory Smad7, but not Smad6. STRAP's interaction with Smad7 exerts a stabilizing effect on Smad7's association with the activated type I kinase receptor which prevents Smad2/3's association and subsequent phosphorylation (Datta and Moses, 2000).
The immunophilin, FKBP12, interacts with the TGF-β type I receptor and acts as a negative modulator of TGF-β function (Wang et al, 1996). It can interact with unactivated type I receptor and functions to stabilize the quiescent receptor state by protecting phosphorylation sites in the GS domain. Upon ligand stimulation, heteromerization and subsequent phosphorylation of the GS domain by the TGF-β type II kinase results in the release of FKBP12 (Chen et al, 1997; Huse et al, 1999). In contrast, the TGF-β type I receptor associated protein-1 (TRAP-1) interacts only with the activated type I receptor kinase (Charng et al, 1998). TRAP-1 is not phosphorylated by the type I kinase and TRAP-1's interaction is reported to have an inhibitory effect on TGF-β signaling. However, a recent report describes a different function for TRAP-1 (Wurthner et al, 2001). In this study, TRAP-1 was found to associate with inactive TGF-β and activin receptor complexes and upon ligand stimulation, TRAP-1 is released. The conformationally altered TRAP-1 is then believed to associate and subsequently chaperone Smad4 to the activated Smad2. The α subunit of ras farnesyl protein transferase (FNTA) preferentially interacts with the activated type I receptor and is considered a substrate because it is phosphorylated by the type I kinase and released thereafter (Kawabata et al, 1995). However the functional significance of this phenomenon remains unexplained. The accessory receptors, endoglin and type III receptor which form heteromeric complexes with the type II receptor have also been reported to modulate TGF-β function. When overexpressed in myoblasts, endoglin inhibited while type III receptor enhanced TGF-β responses (Letamendia et al, 1998). Glycosylphosphatidyl inositol (GPI)-anchored proteins which lack transmembrane and cytoplasmic domains have also been shown to bind TGF-β. These proteins have been identified on certain cell lines (Cheifetz and Massague, 1991), but the identity of these GPI-anchored proteins and the role they may play in TGF-β signaling remain unknown. Recently, the present inventors reported the presence of GPI-anchored TGF-β binding proteins on early passage human endometrial stromal cells (Dumont et al, 1995), human skin fibroblasts (Tam and Philip, 1998) and keratinocytes (Tam et al, 1998). On human keratinocytes, they identified a 150 kDa GPI-anchored TGF-β1 binding protein designated as r150 that can form a heteromeric complex with the types I and II TGF-β receptors (Tam et al, 1998). In addition, they demonstrated that upon hydrolysis fom the cell surface by phosphatidylinositol phospholipase C (PIPLC), the soluble form of r150, retains its ability to bind TGF-β1 in the absence of the types I and II receptors. In addition, it was demonstrated that the GPI anchor is contained in a protein with a molecular weight of 150 kDa (Tam et al, 2001). This novel GPI-anchored TGF-β1 binding protein, r150, has the potential to antagonize or potentiate TGF-β action in keratinocytes. In the absence of the cDNA encoding r150, one way to examine the effect of r150's loss in TGF-β signaling is to enzymatically release the binding protein by PIPLC treatment prior to testing for alterations in TGF-β induced responses. However, the efficacy of exogenously added PIPLC is subject to variability, being affected by pH, temperature, and acylation of GPI-anchored proteins (Shukla, 1982; Chen et al, 1998), thus results obtained may be difficult to interpret. In addition, GPI-anchored proteins that are released may get re-synthesized and re-inserted in the plasma membrane soon after PIPLC hydrolysis. Hence, as an alternative, was have created and isolated a keratinocyte cell line that is mutated in GPI anchor biosynthesis. These cells display a significant loss of r150 from their cell surface, thus allowing a comparative examination of TGF-β mediated cellular responses in the GPI anchor deficient cell line versus the parental HaCat cells under stable experimental conditions
GPI-anchored proteins lack transmembrane and cytoplasmic domains, and are attached to the cell membrane via a lipid anchor in which the protein is covalently linked to a glycosyl phosphatidylinositol moiety. GPI-anchored proteins have been reported to have roles in intracellular sorting (Rodriguez-Boulan and Powell, 1992), in transmembrane signaling (Brown, 1993) and to associate with cholesterol and glycosphingolipid-rich membrane microdomains (Brown and London, 1998; Hooper, 1999). Also, the GPI anchor enables a protein to be selectively released from the membrane by phospholipases (Metz et al, 1994; Movahedi and Hooper, 1997). r150 was characterized as GPI-anchored, based on its sensitivity to phosphatidylinositol phospholipase C (PIPLC). However, it is important to rule out other possibilities, namely, (i) r150 is not itself GPI-anchored, but is tightly associated with a protein that is GPI-anchored, and therefore is susceptible to release by PIPLC; (ii) r150 is a complex of two lower molecular weight proteins which became inadvertently cross-linked during the affinity labeling procedure.
It is now demonstrated that the GPI-anchor is contained in r150 itself and not on a tightly associated protein and that it binds TGF-β1 with an affinity comparable to those of the signaling receptors. Furthermore, the released (soluble) form of this protein binds TGF-β independent of the types I and II receptors. Also, the soluble form inhibits the binding of TGF-β to its receptor. In addititon, we provide evidence that r150 is released from the cell surface by an endogenous phospholipase C. Also, a mutant human keratinocyte cell line with a defect in GPI synthesis was created, which display reduced expression of r150. The results using these mutant keratinocytes suggest that the membrane anchored form of r150 is a negative modulator of TGF-beta responses. These findings, taken together with the observation that r150 forms a heteromeric complex with the signaling receptors, suggest that this accessory receptor in either its membrane anchored or soluble form and its down- or up-regulation may potentiate or antagonize TGF-β responses in human keratinocytes, respectively.
The complete amino acid of a molecule named CD109 was recently disclosed as well as the nucleic acids encoding same (Lin et al. 2002). Sequences comparisons with those of r150 suggest that CD109 is a r150 variant. No definite role has been assigned to CD109 by Lin et al.